The key role of terminators on the expression and post‐transcriptional gene silencing of transgenes

Abstract

Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.

The Whys and Wherefores of Transitivity in Plants

“…For instance, transient expression efficiency of GFP using viral replicons is dramatically improved when multiple introns are added to the reporter sequence ( Marillonnet et al, 2005 ). A similar effect was also observed when an intron-containing GFP transgene was agro-infiltrated in Nicotiana benthamiana leaves, although this effect was dependent on the accompanying terminator sequence ( de Felippes et al, 2020 ). Furthermore, the abundance of RDR6-dependent siRNAs originating from a 35S-driven transgene is reduced when a GFP containing introns is used ( Christie et al, 2011 ).…”

“…During transcription, sequences in the terminator region are recognized by the 3′ end processing machinery resulting in the addition of a poly(A) tail to the nascent transcript and triggering the end of the polymerization process by the RNA polymerase II ( Mandel et al, 2007 ; Kumar et al, 2019 ; Thore and Fribourg, 2019 ). A growing number of studies have shown that different terminators can have a distinct influence in the efficiency of transgene expression ( Ingelbrecht et al, 1989 ; Nagaya et al, 2009 ; Yang et al, 2009 ; Hirai et al, 2011 ; Pérez-González and Caro, 2018 ; de Felippes et al, 2020 ). The improper formation of the mRNA 3′ end has also been implicated with enhanced silencing of transgenes.…”

“…The improper formation of the mRNA 3′ end has also been implicated with enhanced silencing of transgenes. Several members of the polyadenylation machinery have been identified in mutagenesis screens where silencing of GFP was used as a reporter system ( Herr et al, 2006 ; de Felippes et al, 2020 ). Plants carrying mutations in those genes presented increased levels of read-through, which could lead to transcripts missing a poly(A) tail.…”

“…The key function of terminators in avoiding mRNAs being channeled to RDR6 has been made evident in a recent study analyzing the role of different genetic elements in the onset of transgene silencing. Compared to promoters and the presence of introns, terminator usage was the factor that most impacted the production of sRNAs originating from a GFP reporter transgene ( de Felippes et al, 2020 ). Interestingly, the protective role of terminators could be seen even when strong promoters were used, suggesting that adverse effects caused by high levels of transgene expression, as the one induced by the CaMV 35S, could be counter-balanced by strong 3′ regulatory sequences.…”

“…In contrast to organisms supporting transitivity outside of the plant kingdom, the spread of the silencing signal is bi-directional in plants, with secondary sRNAs originating up- and downstream of the target region ( Vaistij et al, 2002 ). Yet, the expansion of the silencing signal in plants shows a clear tendency toward the 3′ region of the transcript ( Braunstein et al, 2002 ; Petersen and Albrechtsen, 2005 ; Bleys et al, 2006b ; Haque et al, 2007 ; Moissiard et al, 2007 ; Aregger et al, 2012 ; Dadami et al, 2014 ; Wroblewski et al, 2014 ; Han et al, 2015 ; de Felippes et al, 2020 ). Another interesting aspect of transitivity in plants is the apparent susceptibility of transgenes in becoming template of RDR6.…”